Download Isolation and Chemical Characterization of a

[CANCER RESEARCH 29, 2034-2038,
November 1969]
Isolation and Chemical Characterization of a Cell-Surface
Sialoglycopeptide Fraction from Novikoff Ascites Cells1
Earl F. Walborg, Jr., Roberta S. Lantz, and Virginia P. Wray
Biochemistry Department, The University of Texas, M. D. Anderson Hospital and Tumor Institute at Houston, Houston, Texas 77025
of the cell surface. This is important since this biologic
characteristic is also associated with the appearance of early
Sialic acid has been shown to contribute significantly to the métastases.
cell-surface charge of tumor cells. In order to elucidate the
Four ionic groups have been shown to contribute to the
chemical nature of the sialic acid-containing molecules present
surface charge of cancer cells: the carboxyl groups of sialic
on the tumor cell surface, a procedure for the isolation of a acid and amino acids, the phosphate group of phospholipids,
Sialoglycopeptide fraction from the surface of Novikoff ascites
and a basic group having a pK of about 10 (5, 9). The sialic
acid carboxyl has received the most attention experimentally
cells has been developed. Digestion of tumor cell suspensions
with papain liberates a Sialoglycopeptide fraction from the cell since glycosidically linked, terminal sialic acid residues can be
surface. This fraction was partially purified using trichlorocleaved from the cell surface by the action of neuraminidase.
acetic acid precipitation,
dialysis, and gel filtration on It has been shown that the anodic mobility of a number of
Sephadex G-50. The Sialoglycopeptide fraction contains
tumors [Ehrlich ascites carcinoma (29); an ascites sarcoma,
65—80%of the neuraminidase-labile sialic acid present on the MC1MAA (29); and polyoma-transformed hamster fibroblasts
surface of these cells. Quantitative analysis of the composition
(12)] is reduced by the action of neuraminidase.
of this fraction revealed the presence of the following
Comparative studies on normal and regenerating liver cells
monosaccharides, expressed per mg glycopeptide: 0.39 /Limole from the adult rat (6, 23) and on embryonic, adult, and
L-strain mouse fibroblasts (14) have indicated that an elevated
sialic acid, 0.41 ¿/moleglucosamine, 0.23 ¿/molegalactosamine,
0.15 ¿/mole mannose, 0.54 jumóle galactose, 0.53 ¿/mole electrokinetic charge density may be associated with growth in
glucose, and 0.15 ¿¿mole
uronic acid. Amino acid analysis
general rather than malignancy in particular. However,
indicated a peptide content of 15%. Amino acids present in Fuhrmann (13) found that a distinct qualitative difference
the highest amounts were threonine, aspartic acid, glutamic
existed between hepatoma cells and the rapidly proliferating
acid, serine, proline, alanine, and valine (molar ratio,
cells of the regenerating rat liver. Proliferating liver cells
4:2:1.5:1:1:1:1).
maintain their electrophoretic
mobility even after neur
aminidase treatment, despite the fact that sialic acid is
INTRODUCTION
released, whereas the mobility of malignant cells is reduced by
more than 50%. This indicates a basic structural difference
Alterations at the cell surface, or cell periphery, have been between the cell surface of cancer cells and their rapidly
implicated in the neoplastic process (1, 11, 28). Evidence for proliferating homologs.
In an effort to ascertain what types of sialic acid-containing
this rests on a wide range of observations on tumor tissue: loss
of contact inhibition (2), altered electrokinetic properties (3), molecules are present at the cell surface, Langley and Ambrose
decreased intercellular
communication
as measured by (15, 16) have isolated and partially characterized a Sialo
junctional membrane resistance (17, 18), and decreased
glycopeptide
fraction,
obtained
by digesting Ehrlich
incidence of the tight junction (11).
ascites cell suspensions with trypsin. It is important to extend
Increased anodic electrophoretic mobility is exhibited by such studies to other tumor systems and to normal
several different lines of tumor cells, e.g., diethylstilbestrolhomologous cells. This paper describes the isolation and partial
induced hamster renal tumor (3), Ehrlich ascites carcinoma
characterization of a Sialoglycopeptide fraction isolated from
(24), and polyoma-transformed
hamster fibroblasts (12).
Novikoff ascites tumor, a rat hepatoma originally induced by
4-dimethylaminoazobenzene
(20).
Purdom et al. (22) have shown that the selection of a biologic
characteristic, namely, the ease of producing the ascites form,
MATERIALS AND METHODS
is correlated with a progressive increase in the negative charge
SUMMARY
'"This research was supported by Research Grant P-451 from the
American Cancer Society and USPHS Institutional Grant No. FR
05511-03-5.
Received March 3,1969; accepted April 17,1969.
2034
Collection of Tumor Cells. The Novikoff ascites tumor was
maintained in Sprague-Dawley rats, 7—9weeks of age. At 5—7
days after inoculation, ascites fluid was collected and diluted
with 3 volumes of the following buffer: 0.105 M NaCl, 4.8
mM KC1, 5.0 mM glucose, 0.91 mM NaH2PO4, and 10.9 mM
Na citrate, adjusted to pH 6.5 (Buffer 1). All washes of the
CANCER RESEARCH VOL. 29
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Cell-Surface Sialoglycopeptide
cells utilized buffer chilled to 4°C.Twenty-five-mi portions of
solution was allowed to stand at 23°Cfor about 18 hr. The
this suspension were distributed to 30-ml graduated Kolmer
centrifuge tubes (Corning No. 8380). In order to remove
contaminating red blood cells, the cell suspensions were
centrifuged at 10 X g for 20 min. After removal of the
supernatant fluid by aspiration, the cells were resuspended in
20 ml of the same buffer and were centrifuged for 15 min at
lOXf.
Neuraminidase
Digestion. The twice-washed cells were
resuspended in 20 ml of the following buffer: 0.116 M NaCl,
40 mM Na acetate, and 4.6 mM CaCl2, adjusted to pH 5.5
(Buffer 2). The cells were centrifuged for 15 min at 10 X g.
The supernatant fluid was aspirated, and the cells (approx. 3
ml) were resuspended in 19.5 ml of the same buffer. The cell
suspension was incubated with agitation at 37°Cfor 10 min
precipitate was removed by filtering through Whatman No. 42
filter paper and discarded. The TCA was extracted from the
filtrate with 1 volume, •§
volume, and -j volume of ethyl
ether. Insoluble material, present at the interface, was removed
with the ether washes. The ether-extracted
solution was
filtered, adjusted to pH 6 with dilute NaOH, and dialyzed
against four changes of deionized water. The dialyzed material
was concentrated by flash evaporation to 10 ml. An aliquot of
the concentrated sample was analyzed for sialic acid by the
method of Warren (31), and the remainder of the sample was
submitted to gel filtration on a 3 x 50 cm column of Sephadex
G-50, using water at a flow rate of 20 ml/hr as eluent. Five-mi
fractions were collected. The sialic acid-containing fractions
(effluent volume 100-200 ml) were pooled and lyophilized.
using a Warner-Chilcott shaker bath, Model 2156. Following
this preincubation, 0.5 ml of neuraminidase solution (from
Vibrio cholera strain Z4, 500 units/ml, Mann Research
Laboratories, New York, N. Y.) was added. As controls, cells
were incubated without enzyme, and neuraminidase was
incubated in Buffer 2 without cells. Packed cell volumes were
determined by centrifuging cell controls for 10 min at 250 X
g. At various time intervals, 3-ml aliquots of each incubation
were removed, chilled in an ice bath, and the cells centrifuged
at 250 X g for 10 min. A 2-ml aliquot was taken from the
supernatant liquid. To this was added 0.3 ml of 100%
trichloracetic acid (TCA) to precipitate the enzyme. The
precipitate was removed by centrifugation for 15 min at 500 X
g. Aliquots (0.5 ml) of the supernatant liquid were taken for
analysis of sialic acid by the method of Warren (31).
Papain Digestion. The twice-washed cells were resuspended in
20 ml of the following buffer: 0.105 M NaCl, 4.8 mM KC1.5.0
mM glucose, 0.91 mM NaH2P04l 10.9 mM Na citrate, 5 mM
cysteine, and 5 mM Versene, adjusted to pH 6.5 (Buffer 3).
The cells were centrifuged for 15 min at 10 X g. The
supernatant fluid was aspirated, and the cells (2—3ml) were
resuspended in 19 ml of the same buffer. The cell suspensions
were incubated with agitation for 10 min at 37°C.Following
this preincubation,
1 ml of a papain suspension (2 times
crystallized, 350-600 units/ml, Worthington Biochemical Co.,
Freehold, N. J.) was added to each flask, and the incubation
was continued for 40 min. As controls, cells were incubated
without enzyme, and papain was incubated in Buffer 3
without cells. The incubation flasks were swirled manually
every 10 min. After 40 min the incubation mixtures were
chilled in ice and centrifuged in 30-ml graduated Kolmer
centrifuge tubes for 10 min at 250 X g. The packed volume of
cells (approx. 1 ml/incubation) was recorded. The average
packed volume of the cell controls was used to calculate the
cell volumes for each experiment. The packed volume of the
papain-treated cells was usually the same as the cell controls
and in no cases exceeded a 15% increase in packed volume.
Cell counts have been performed after 40 min of digestion
with papain, and no loss of cells could be shown to occur. One
ml of packed cells is equivalent to 1.7 to 2.0 X IO8 cells.
Preparation of Crude Glycopeptide Fraction. The super
natant fluid obtained from the papain-treated cells was
collected. An equal volume of 100% TCA was added, and the
NOVEMBER 1969
Analysis of Column Effluents. The UV absorbance was
measured utilizing the Gilford Model 220 spectrophotometer.
Neutral sugar was determined by the method of Dubois étal.
(10), using D-galactose (Mann Assayed Grade, Mann Research
Laboratories, New York) as a standard. After hydrolysis (2 N
HC1, 100°C, 8 hr) hexosamine was determined by the
Elson-Morgan reaction, as described by Antonopoulos et al.
(4) using D-glucosamine-HCl (Mann Assayed Grade) as a
standard. Sialic acid was quantitated using the method of
Warren (31). 7V-acetylneuraminic acid (NANA), obtained from
Pierce Chemical Co., Rockford, 111.,was used as a standard.
Prior to sialic acid analysis, aliquots of the column fractions
were dried and hydrolyzed in 0.5 ml of 0.1 N H2SO4 for 1 hr
at 80°C.
Quantitative Analysis of the Amino Acid and Carbohydrate
Components of the Isolated Sialoglycopeptide Fraction. Ten
mg of the Sialoglycopeptide fraction were dried in vacuo over
P2O5 at 100°Cin a drying pistol (Corning No. 3690), and the
dry material was weighed on a microanalytical balance to an
accuracy of ±20Mg- This material was dissolved in a 10-ml
volumetric flask, and aliquots were taken for analysis. Amino
acid analysis was performed utilizing the Beckman Model 120
B amino acid analyzer following hydrolysis in constant boiling
HC1 for 20 hr at 110°C under N2 at a glycopeptide
concentration of 4 mg/ml. Separation and quantitation of
hexosamines was accomplished using the Beckman Model B
amino acid analyzer, according to the method of Walborg et al.
(26), following hydrolysis in 2 N HC1 for 8 hr at 100°Cunder
N2 at a glycopeptide concentration of 1 mg/ml. Separation
and quantitation of the neutral sugars was performed using the
ion-exchange, Chromatographie procedure of Walborg et al.
(27), following hydrolysis in l N H2S04 for 8 hr at 100°Cat a
glycopeptide concentration of 4 mg/ml. Sialic acid, expressed
as NANA, was measured by the method of Warren (31),
following hydrolysis in 0.1 N H2SO4 at 80°Cfor 1 hr. Uronic
acid, expressed as glucuronic acid, was quantitated utilizing
the colorimetrie method of Bitter and Muir (7). In order to
correct for nonspecific
interference
from other sugar
components present in the Sialoglycopeptide fraction, a
solution containing hexosamine, neutral sugar, and sialic acid
in an amount
equivalent
to that
present
in the
Sialoglycopeptide fraction analyzed, was included as a control.
2035
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Earl F. Walborg, Jr., Roberta 5. Lantz, and Virginia P. Wray
RESULTS
Determination
of the Neuramin¡dase-labile, Cell-Surface
Sialic Acid. The release of sialic acid from cell suspensions of
Novikoff ascites cells is shown in Chart 1. The results are
calculated as jumólesialic acid, expressed as NANA, per ml of
packed cells. Using the average of the 90- and 120-min points
on the release curves, the neuraminidase-labile sialic acid was
0.26 ±0.01 S.D. pinole NANA/ml cells. A two-fold increase in
the neuraminidase concentration did not significantly increase
the amount of sialic acid released.
0.3NEURAMINIDASE
speeds following incubation with papain. If cell lysis occurs
during papain incubation, nucleic acids are released into
solution, increasing the viscosity and making it impossible to
pack the cells at low centrifugal force. The agreement of the
packed volumes of cells for both the papain-digested and
control cells indicated that cell lysis did not occur in the
method described herein.
The elution pattern obtained by gel filtration on the
TCA-precipitated,
ether-extracted,
dialyzed, concentrated,
Sialoglycopeptide fraction is shown in Chart 2. This sample
represents the Sialoglycopeptide fraction obtained from 5.4 ml
of packed Novikoff ascites cells. Larger amounts of
Sialoglycopeptide, representing 18—30ml of packed cells, were
fractionated by gel filtration. These preparative scale columns
were analyzed for UV absorbance and sialic acid (31), and the
sialic acid-containing fractions were pooled and lyophilized.
Recovery of sialic acid from the column was quantitative. The
yield of crude Sialoglycopeptide was 250—450 fig per ml of
packed cells.
NOVIKOFF
1.0-
TUMOR
CELLS +
PAPAIN
NANA
HEXOSAMINE
U V Abs.
0.50-
.+
Vi
I
30
60
120
0.5-
0.25-
Minutes of Incubation
Chart 1. Rate of release of sialic acid from Novikoff ascites cells on
incubation with neuraminidase. Sialic acid (NANA) was assayed by the
method of Warren (31). The packed cell volumes were determined by
centrifugation for 10 min at 250 X g.
!\ ÃŽ
v".
i»
t
Isolation of the Sialoglycopeptide Fraction. Sialic acid
analysis of the TCA-precipitated, ether-extracted, dialyzed,
concentrated Sialoglycopeptide fraction indicated that papain
released 0.20 ±0.03 S.D. /umole sialic acid, expressed as
NANA, per ml of packed cells. These values were obtained
from five separate preparative scale isolations of Sialoglyco
peptide, each of which utilized 40-rnin incubations with
papain. Incubation for 60 min released the same amount of
Sialoglycopeptide, indicating that maximal release had been
attained by incubation for 40 min. Approximately 65-80% of
the neuraminidase-labile sialic acid was released by papain in
the form of a Sialoglycopeptide.
Since ascites fluid contains glycoproteins which may be a
source of glycopeptides (16), three washes of the cells were
performed prior to enzyme digestion. Inclusion of an
additional wash in the procedure did not reduce the amount of
sialic acid released by papain, indicating that soluble
glycoproteins had been removed. Cell lysis during papain
digestion was minimal as evidenced by cell counts and by the
ability to centrifuge the cells at relatively low centrifugal
2036
TUMOR
0.2-
0.10-
0.1-
0.05-
CELL CONTROL
PAPAIN
100
EFFLUENT
200
VOLUME,
I,
,' V,
CONTROL
300
ml
Chart 2. Gel filtration of the Sialoglycopeptide fraction from the cell
surface of Novikoff ascites cells utilizing a 3 x 50 cm column of
Sephadex G-50, operated at a flow rate of 20 ml/hr with water as
eluent. The effluent was assayed for sialic acid by the method of
Warren (31), following hydrolysis in 0.1 N H2SO4 for 1 hr at 80°C.
Hexosamine was quantitated by the Elson and Morgan reaction (4),
following hydrolysis in 2 N HC1 for 8 hr at 100°C.The volume outside
the gel (V0) was determined by measuring the effluent volume of Blue
Dextran 2000, obtained from Pharmacia Fine Chemicals, Inc. The sum
of the volume inside the gel (V¡)and the V0 was determined by
measuring the effluent volume of chloride ions. NANA,
JV-acetylneuraminic acid.
CANCER RESEARCH VOL. 29
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Cell-Surface Sialoglycopeptide
Quantitative Analysis of the Amino Acid and Monosaccharide Components of the Crude Sialoglycopeptide Fraction.
The results of these analyses are shown in Table 1.
Ion-exchange chromatography of the neutral monosaccharides,
as their borate complexes, indicated the presence of mannose,
galactose, and glucose. A component, eluted at a relative
retention volume consistent with fucose, was also observed.
This component, present at a concentration of 0.05 jumole/mg
glycopeptide, accounts for only 0.7% of the weight. Because
of the low concentration present, additional analyses on larger
quantities of material will be required to substantiate the
presence of fucose. The amino acids present in the highest
amounts were threonine, aspartic acid, glutamic acid, serine,
proline,
alanine,
and valine, the molar ratio being
4:2:1.5:1:1:1:1.
Total recovery of the weight of the material
was approximately
60%, The UV absorbance spectrum
exhibited a shoulder at 270 m;u. The optical density of a 1%
solution of the Sialoglycopeptide fraction, measured in a 1-cm
optical path at 260 mn, was 5.23, indicating that
contamination with nucleic acid was minimal.
(65-80%) of the neuraminidase-labile sialic acid was released
by this procedure.
The Sialoglycopeptide fraction isolated from Novikoff ascites
cells undoubtedly represents a family of glycopeptides. The
elution profile, obtained by gel filtration, shows that the ratio
of sialic acid:hexosamine:neutral
sugar varies with effluent
volume, indicating a multiplicity of components. Further
purification of this Sialoglycopeptide fraction is under way
prior to undertaking structural studies. Since amino acids and
carbohydrates account for only 60% of the weight of the
Sialoglycopeptide, the possible presence of other components
is being pursued.
Langley and Ambrose (15, 16) have isolated a Sialoglyco
peptide fraction from Ehrlich ascites carcinoma by digestion
of cell suspensions with trypsin. The Sialoglycopeptide fraction
liberated by trypsin was further degraded by digestion with
pronase. Comparison of their analytical data with that
obtained for the Novikoff ascites cell Sialoglycopeptide
fraction reveals several differences. The relative quantities of
acidic and hydroxyl amino acids are of interest since these
amino acids have been implicated in the linkage between the
peptide and carbohydrate moieties. The Ehrlich Sialoglyco
peptide fraction has a predominance of acidic amino acids (7
1ComponentPeptideCarbohydrateSialic
Table
moles of acidic to 2 moles of hydroxyl amino acids), whereas
(%)15.611.48.24.72.48.88.6
the Novikoff Sialoglycopeptide
fraction contains more
hydroxyl amino acids (5 moles of hydroxyl to 3.5 moles of
acidic amino acids). Neutral sugar is an important constituent
acidaGlucosamine^Galactosamine''MannoseGalactoseGlucoseUronic
of the Novikoff Sialoglycopeptide fraction, whereas the
Ehrlich Sialoglycopeptide fraction contains no neutral sugar.
Hexosamine and sialic acid are present in equimolar quantities
in the Ehrlich Sialoglycopeptide fraction, whereas the ratio of
hexosamine to sialic acid is 1.6 in the Novikoff Sialoglyco
acidMmole/mg0.390.410.230.150.540.53<0.16Residue
peptide fraction.
The Novikoff Sialoglycopeptide fraction is similar in several
Total
59.7
respects to the glycopeptides released by trypsin from intact
human erythrocytes, described by Winzler et al. (30). These
Chemical composition of the Sialoglycopeptide fraction obtained
from Novikoff ascites tumor.
glycopeptides contain neutral sugar, primarily galactose, and
"Expressed as jV-acetylneuraminic acid.
are rich in serine and threonine.
''Expressed as jV-acetylhexosamine.
Abercrombie and Ambrose (1) have proposed that alterations
at the cell periphery contribute significantly to many of the
manifestations of the malignant cell: invasiveness, liability to
DISCUSSION
passive dissemination, and disorganization of structure. The
investigation of the cell-surface sialoglycopeptides of rat
Trypsin has been widely used to release sialoglycopeptides
hepatomas and their normal homologous tissue will allow
from the cell surface (8, 9,15,21,25,
30).Papain was chosen
chemical alterations to be correlated with normal and
for use in these experiments because it hydrolyzes a broader
neoplastic growth processes.
spectrum of peptide bonds (25). Because of its specificity,
more cleavage sites on the lipoglycoprotein subunits of the
plasma membrane should be available to this enzyme. Also, its ACKNOWLEDGMENTS
broad specificity should enable more complete hydrolysis of
The authors gratefully acknowledge the support and interest of Dr. A.
the peptide moiety of the released glycopeptides, thereby
C. Griffin, Dr. Robert B. Huribert, and Dr. D. N. Ward. Cell counts
minimizing differences in the molecular weight of the
were performed by Miss Oleta Klatt of the Department of Surgery.
glycopeptides due to varying peptide chain lengths. The
availability of this enzyme in highly purified form eliminates
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Earl F. Walborg, Jr., Roberta S. Lantz, and Virginia P. Wray
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Isolation and Chemical Characterization of a Cell-Surface
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Earl F. Walborg, Jr., Roberta S. Lantz and Virginia P. Wray
Cancer Res 1969;29:2034-2038.
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